The cofactor challenge in synthetic methylotrophy: bioengineering and industrial applications.

Methanol is a promising feedstock for industrial bioproduction: it can be produced renewably and has high solubility and limited microbial toxicity. One of the key challenges for its bio-industrial application is the first enzymatic oxidation step to formaldehyde. This reaction is catalysed by methanol dehydrogenases (MDH) that can use NAD+, O2 or pyrroloquinoline quinone (PQQ) as an electron acceptor. While NAD-dependent MDH are simple to express and have the highest energetic efficiency, they exhibit mediocre kinetics and poor thermodynamics at ambient temperatures. O2-dependent methanol oxidases require high oxygen concentrations, do not conserve energy and thus produce excessive heat as well as toxic H2O2. PQQ-dependent MDH provide a good compromise between energy efficiency and good kinetics that support fast growth rates without any drawbacks for process engineering. Therefore, we argue that this enzyme class represents a promising solution for industry and outline engineering strategies for the implementation of these complex systems in heterologous hosts.

NADPH-Auxotrophic E. coli: A Sensor Strain for Testing in Vivo Regeneration of NADPH.

Insufficient rate of NADPH regeneration often limits the activity of biosynthetic pathways. Expression of NADPH-regenerating enzymes is commonly used to address this problem and increase cofactor availability. Here, we construct an Escherichia coli NADPH-auxotroph strain, which is deleted in all reactions that produce NADPH with the exception of 6-phosphogluconate dehydrogenase. This strain grows on a minimal medium only if gluconate is added as NADPH source. When gluconate is omitted, the strain serves as a "biosensor" for the capability of enzymes to regenerate NADPH in vivo. We show that the NADPH-auxotroph strain can be used to quantitatively assess different NADPH-regenerating enzymes and provide essential information on expression levels and concentrations of reduced substrates required to support optimal NADPH production rate. The NADPH-auxotroph strain thus serves as an effective metabolic platform for evaluating NADPH regeneration within the cellular context.

Artificial pathway emergence in central metabolism from three recursive phosphoketolase reactions.

The promiscuous activities of a recursive, generalist enzyme provide raw material for the emergence of metabolic pathways. Here, we use a synthetic biology approach to recreate such an evolutionary setup in central metabolism and explore how cellular physiology adjusts to enable recursive catalysis. We generate an Escherichia coli strain deleted in transketolase and glucose 6-phosphate dehydrogenase, effectively eliminating the native pentose phosphate pathway. We demonstrate that the overexpression of phosphoketolase restores prototrophic growth by catalyzing three consecutive reactions, cleaving xylulose 5-phosphate, fructose 6-phosphate, and, notably, sedoheptulose 7-phosphate. We find that the activity of the resulting synthetic pathway becomes possible due to the recalibration of steady-state concentrations of key metabolites, such that the in vivo cleavage rates of all three phosphoketolase substrates are similar. This study demonstrates our ability to rewrite one of nature's most conserved pathways and provides insight into the flexibility of cellular metabolism during pathway emergence.